Hepatitis C virus (HCV) exacts a heavy toll on global health by causing acute and chronic hepatitis, cirrhosis, as well as hepatocellular carcinoma. Paradoxical to this cold reality, there is no vaccine available to prevent the infection, and combination therapy with pegylated IFN-a and ribavirin is only effective in 40-80% of patients and has severe side effects. Fortunately, with the recently developed infectious clones, we are finally able to dissect the entire viral life cycle at the molecular level. It is conceivable that novel antiviral therapies and treatment regimens relieving HCV associated pathologies will become available within foreseeable future given the accelerated pace of research. As far as what this application concerns, accumulating evidence has highlighted the importance of tight junction (TJ) in HCV entry. Specifically, we and others reported that the tight junction protein occludin (OCLN) is indispensible for HCV envelope protein-dependent entry. Evidence also indicated that OCLN partially accounts for the narrow host range of HCV. However, murine cells expressing all known human (co-)receptors remained resistant to cell culture grown HCV (HCVcc) infection, suggesting either additional human factors are required or dominant inhibitors exist in murine cells. Interestingly, we observed that both OCLN and claudin-1 were downregulated in HCVcc-infected hepatoma cells. Subsequent study suggested that OCLN is increasingly cleaved in HCV infected cells. In light of these findings, we believe novel therapeutics, novel infection systems, and novel insights in understanding HCV-associated pathogenesis may be developed. We hypothesize that antibody against OCLN extracellular loops or OCLN-derived peptide may block the spreading of virus (to be tested in Aim 1); additional host factors are required to support optimal HCV infection in murine cells or restriction factors exist in these cells (to be tested in Aim 2); downregulation of TJ proteins during HCV entry may alter cell physiology (to be tested in Aim 3). We plan to test the hypotheses by (i) assessing the effect of antibodies against OCLN or OCLN-derived peptides on HCV entry. (ii) identifying host factors promoting or restricting HCV infection of murine cells. (iii) analyzing the impact of HCV infection on TJ integrity and hepatocyte functions.